The Effects Of Inflammatory Mediators IL-1β And IL-3on Endothelial Cells Expressing The Lymphatic Phenotype

Background:Lymphatic system is an significant auxiliary system in cardiovascular circulatory system for its critical role in the regulation of the osmotic pressure, environmental stability, immunity and inflammation. There is an intimate correlation among lymphangiogenesis and many pathophysiological processes, especially in inflammation, embryonic development, wound healing and tumor metastasis. So exploring new mechanism for the lymphangiogenesis and treatment of diseases is of great significance.It was believed in the previous researches that there were two major routes in the lymphangiogenesis:the new born lymphatic vessels were budding from the intrinsical lymphatic vessels with VEGF-C, and the endothelial progenitor cells in peripheral blood could be induced by VEGF-C differentiating into lymphatic endothelial cells (LECs). Recent studies indicated that many inflammatory mediators had important contact with lymphatic markers expression and lymphangiogenesis, such as LPS, TNF-a, IFN-γ, IL-1, IL-3, IL-6and so on. However, the inflammatory mediators regulating mechanism of lymphatic endothelial phenotype is still unclear. It was reported that inflammatory mediators IL-1β or IL-3may induce endothelial cells to express lymphatic markers through NF-κB p50pathway; while there were some reports argue that IFN-y/TNF-a prestimulation was additive to subsequent IL-3induced Prox-1and Podoplanin expression in BECs. IL-1plays an important part in many pathological processes. So, we raise a question that whether can IL-1induce BECs to express lymphatic endothelial phenotype by NF-κB pathway that is, inducing BECs transdifferentiating into LECs? There was few relevant report. This article focuses on the regulating role of IL-1β and IL-3in inducing BECs turned into LECs.Objective:To invest the induction or maintenance of mechanisms and critical elements about lymphatic endothelial phenotype through IL-3and IL-1(3stimulating HUVECs and the cell line CRL-1730separately.Methods:Firstly, primary HUVECs were obtained and cultured as described before. The cells were immunostained with antibody to vWF to determine whether they were vascular endothelial cells. Then, the cells were stimulated with IL-1β/IL-3after24h, recording their morphology changes when they confluence. With immunocytochemistry and RT-PCR, the lymphatic markers expression were detected, we tested themselves after the stimulation of IL-1β or IL-3separately for24h. Then, we used the CRL-1730cells insteading for the cultured HUVECs taken on all above experiments. Moreover, we compared the treated with IL-1β or untreated cell line with the untreated primary HUVECs about the amout of the differences in expressing the lymphatic markers by Real-time PCR. At last in the presence of specific inhibitors of NF-κB (PDTC) for1h, we detected the expression of lymphatic markers before adding IL-3for a certain time.Results:Immunocytochemical and RT-PCR both indicated that the cultured HUVECs did expressed lymphatic markers while the CRL-1730cells did not express. But after the IL-3stimulation, HUVECs had changed their shape from pebbles to spindle, and test showed that they were positive, the inhibition group was negative. The CRL-1730cells which was treated with IL-1β group was upregulated by Real-time PCR detection. And this also verifed the result of RT-PCR:the untreated primary HUVECs group in the amount of lymphatic markers was much higher than the untreated CRL-1730cells.Conclusion:Firstly, both inflammatory mediators IL-1(3and IL-3could induce BECs to express lymphatic endothelial phenotype; secondly, IL-1β may induce BECs to express lymphatic phenotype through the NF-κB pathway. Signification:We would like to study the inflammatory mediators in the role of inducing BECs transdifferentiating into LEC, and explain the importance of mechanism of lymphangiogenesis. For the treatment of lymphangiogenesis and related disorders, we may build the basis.